Resolved
Authors: Michael Zeisberg
Journal: Journal of the American Society of Nephrology (2010)

Epithelial-mesenchymal transition (EMT) was originally described as an early embryonic event enabling primitive epithelia to migrate as mesenchymal cells forming the mesoderm and for primitive neuroepithelia to move as neural crest cells.1 Without the mechanism of EMT, complex organisms could not exist. It is also without argument that EMT operates in adult cancer cells as a molecular program for tumor invasion and metastasis.2–4 The cellular evidence for EMT was first described by Greenburg and Hay5 in the early 1980s in the context of embryonic development. This same group then reported their original embryonic EMT criteria also applied to adult kidney tubular epithelial cells acquiring a fibroblast phenotype upon immersion in collagen gels in vitro.6 Today, to address the variations of EMT seen in diverse biologic contexts, EMT involving embryonic epithelia is now classified as type 1 EMT; type 2 EMT refers to adult epithelial and endothelial cells transitioning to fibroblasts, and EMT involving metastatic cancer cells is called type 3 EMT.1,7,8 The debate question posed here is quite simple: Can the activation of the same pathways that cause EMT in embryonic epithelia9 and cancer10 cells also cause adult somatic cells (epithelia and endothelia) to acquire a fibroblast phenotype? In taking the affirmative position in this debate, there is an extensive body of evidence demonstrating EMT in the setting of fibrogenesis; induction of EMT transcriptional programs channel both adult epithelial and endothelial cells to acquire a mesenchymal or fibroblast phenotype. A PubMed search revealed the contribution of EMT to fibrogenesis is currently supported by more than 300 original publications and opposed by five or so that failed to detect EMT in humans or experimental models of tissue fibrosis,11–15 the latter issue perhaps initiating this debate. The notion that adult somatic cells can undergo EMT has evolved greatly in the past 15 years. Although type 2 EMT contributes to fibroblast accumulation in various fibrotic tissues, including lung, skin, eye lens, intestine, liver, and peritoneum, I largely confine my comments to its role in kidney fibrosis. The current view of EMT in renal fibrosis is that stimuli from an inflammatory microenvironment induce conserved signaling pathways in epithelial and endothelial cells that also regulate EMT in embryonic epithelia and cancer cells.8 Adult tubular epithelial cells respond by acquisition of a mesenchymal phenotype, which in its most complete form leads to formation of a fibroblast.7 When detection of EMT in the context of fibrosis is discussed, a few basic principles must be understood to avoid misconception: First, EMT, in its most extreme form, can lead to formation of a fibroblast, but the transition does not necessarily have to go that far. This means that the number of epithelial cells undergoing EMT does not necessarily equal the number of cells that become fibroblasts. In fact, the exact percentage of tubular epithelial cells or renal endothelia that become fibroblasts in a diseased kidney is entirely unknown. Second, there is consensus that fibroblasts within fibrotic tubulointerstitium proliferate—irrespective of their origin.16–18 This means, hypothetically, substantial numbers of fibroblasts may originate from only a few EMT-derived fibroblasts over prolonged periods of time. Third, EMT is a dynamic and reversible process,19 suggesting there are intermediate stages whereby epithelial or endothelial cells display features of their parental origin as well as those of an emerging fibroblast, and suggesting there are advanced stages by which point epithelial cell characteristics have been lost. Thus, intermediate stages of EMT precede advanced stages during renal fibrosis, making the timing of tissue analysis critical for interpretation. To detect EMT in vivo, one would like to view epithelial cells transitioning into fibroblasts and migrating to the interstitium in real time. Unfortunately, this is not feasible with current technology. Instead, one is left with compelling snapshots of the transitional process through histochemical analysis of fibrogenic tissue.20,21 Most studies center around co-immunostaining for markers of epithelial cell lineage, fibroblast markers, and activation of the EMT transcriptional program, particularly nuclear translocation of Snail, Twist, β-catenin, or CBF-A.7,22 Detection of intermediate stages of EMT in injured kidney is straightforward and is the current gold standard; one example is a tubular epithelial cell that expresses the fibroblast marker FSP1, in which Snail signaling is active and E-cadherin is lost.22,23 Detection of near-complete EMT is more complex because cells of epithelial or endothelial origin now look phenotypically like fibroblasts. For proving EMT at this stage, original parental cells must express a stable tag to identify its epithelial or endothelial origin irrespective of current phenotype.24 Ten studies of mice using different lineage-tracing transgenes in epithelial or endothelial cells showed EMT25–34; three of those studies were in kidney.26,27,33 These advanced stages of type 2 EMT cannot be detected within fibrotic human kidneys, because human epithelial cells do not carry obvious lineage tags; an α-smooth muscle actin–positive myofibroblast, for example, provides no known clues about its origin; however, a number of indirect studies suggested that EMT is operative in human tissue.35–37 To use current technology to its best capacity to detect EMT, I suggested previously that a combination of epithelial cell markers, possible lineage tags, fibroblast markers, and transcription signals, best establish the presence of EMT in vivo.7 Adhering to such criteria, hundreds of research articles reported evidence for EMT in various fibrotic tissues, including exquisite detail regarding the biochemistry of transitional signaling.38 Many of these studies identified transcription factors that unequivocally induce EMT in adult epithelial cells (Snail, Twist, β-catenin, CBF-A),22,39 as they do in embryonic epithelia and cancer cells.4 In fact, induction of a Snail transgene alone is sufficient to induce EMT and recapitulate all stages of kidney fibrosis in mice.39 Short of a movie, there is abundant documentation that during renal fibrogenesis, EMT is initiated in single cells within tubular cross-sections.20,21,27 Activation of this EMT program is soon followed by expression of fibroblast markers associated with loss of contact with tubular basement membrane.20 These initial EMT stages have been captured not only in mice27 but also in human renal biopsies.37,40–42 Lineage-tracing experiments also allow for detection of tubular epithelial cell–derived26,27 and endothelial cell–derived fibroblasts within the kidney43 as well as in other organs with fibrosis.26,28,30,31,34 Most important, our advances in understanding EMT have produced several effective strategies to treat experimental fibrosis in rodents. Molecules such as bone morphogenic protein 719 and hepatocyte growth factor44 inhibit EMT and are leading candidates for clinical development in treating chronic kidney disease. A few studies, however, recently came to the conclusion that EMT may not be operative in the context of fibrogenesis.11–13 Here, I attempt to understand why studies with almost identical experimental setup lead to opposite results. As it pertains to the kidney, a recent study by Humphreys et al.11 induced renal fibrosis after unilateral ureteral obstruction in kidneys marked with the Six2.cre promoter that should be active in most renal epithelium. Unlike numerous previous publications that unequivocally demonstrated expression of fibroblast markers by intermediate-stage tubular epithelial cells in various models of chronic kidney fibrosis (unilateral ureteral obstruction,45–47 nephrotoxic serum nephritis,19 5/6 nephrectomy,48 streptozotocin-induced diabetic kidneys,49 anti–tubular basement membrane nephritis,50 chronic allograft nephropathy,51 polycystic kidney disease,52 and HIV-associated nephropathy53), this study failed to detect α-smooth muscle actin–positive or FSP1+ immunostaining in a single epithelial cell in vivo. Predictably, this study then also failed to detect advanced stages of EMT forming FSP1+ fibroblasts as assessed by dsRed double-labeling experiments in ACTB-Bgeo, DsRed.MST double-transgenic reporter mice. On the basis of these data, which were collected in one single disease model, this study tried to challenge the existence of both the intermediate and advanced stages of EMT—in contrast to positive findings in more than 100 original publications on various kidney injury models.54 The complete failure to detect EMT markers even in a single tubular epithelial cell in vivo is puzzling and difficult to interpret from the outside. Attempts to find biological explanations for the discrepancies to previous studies are also hindered because no information is provided for determination of whether EMT transcriptional pathways were activated in this particular set of experiments. Furthermore, no technical advances that provide explanation for why so many previous studies would be in error were made. With regard to expression of fibroblast markers, this group even failed to find collagen I–producing FSP1+ cells in the kidney at all,55,56 which goes against more than 60 articles in the literature and may be the first of its kind; most experimentalists who stain for FSP1 in tissue use antigen retrieval technology before immunochemistry, which was not done here. Furthermore, because expression of collagen I was used as their gold standard for defining fibroblasts, this failure to detect was used as further evidence against EMT. I object to this definition of a fibroblast based solely on collagen I expression because not all fibroblasts produce collagen all of the time. Another issue in this study is the collagen I–GFP reporter gene they used was originally optimized to detect collagen-producing stellate cells in the liver. There currently is no collagen I promoter construct available that mirrors type I collagen expression in all cell types,57 and the efficacy of this particular construct to detect type I collagen expression in kidney fibroblasts was not assessed, possibly explaining why they could not detect a single collagen-producing FSP1+ fibroblast.55,56 Despite my concerns about this negative experiment, as well as the others on which I have not commented, I welcome this debate because it not only highlights where misunderstandings of EMT have led to confusion but also pinpoints where further advances can be made. I understand that EMT is sometimes met with skepticism because it cannot be watched to evolve before an observer's eye; however, neither are leukocytes that undergo diapedesis through vessel walls or cancer cells that metastasize into the bloodstream. Only few will doubt these latter events occur even though they are just as difficult to observe as EMT. Science should evolve through open discussion, but for a fruitful discussion there should be at least some common agreement, in this case, what is EMT, how it should be detected, and what is a fibroblast—questions that have largely been addressed in the past decade using sophisticated genetic and biochemical approaches to which we should adhere. Conceptually, induction of EMT makes a lot of sense for the kidney as a protective mechanism. Most transcriptional pathways in the EMT program (Snail, CBF-A, and Twist) are also known to be anti-apoptotic and hence provide a way for tubular epithelial cells to avoid imminent death.1 Because tubulointerstitial injury is associated with disturbed interaction of tubular epithelial cells with their tubular basement membrane,20 watershed cells are also at risk for falling off into their lumens.46,42 The migratory capacity provided by EMT enables these transitional cells to invade the basement membrane20 and evade this threat.58 Furthermore, EMT from a fibroblast-centric view is a highly efficient way to recruit fibroblasts rapidly to local sites of tubulointerstitial injury as opposed to their recruitment from more distant sites in tissue or even from the bone marrow using fibrocytes that migrate through the bloodstream.59 The advantageous economy of fibroblast recruitment by EMT is even more obvious at sites with more pronounced surfaces (e.g., skin, epicardium), where EMT is an essential process to seal new lesions.60,61 Why would adult kidney epithelial cells not also undergo EMT under duress? As with Occam's Razor, it just makes sense. Disclosures None. M.Z. is funded by research grants R01DK081576, R03DK081687, and K08DK074558 from the National Institutes of Health and research funds from the Beth Israel Deaconess Medical Center for the Division of Matrix Biology.